The incredible clinical and commercial successes of recombinant protein therapeutics cemented the use of mammalian cells as the premier production hosts for these products. However, we can further exploit these cells to harness their potential for addressing current and future medical needs through metabolic and advanced engineering of these cells. To do so, we need a deeper understanding of the intricate gene regulation network that governs these cells and the ability to attain precise control of gene expression levels. In addition, some of these applications, such as gene therapy and immunotherapy, could benefit greatly by refraining from using viral-derived genetic elements. Therefore, this work seeks to establish additional transcriptional control elements to improve our ability to regulate expression with generalizable approaches and methods, facilitating the adaptation of these techniques for any mammalian cell type of interest. Here, we successfully demonstrated three key genetic elements can be utilized to tune gene expression in a rational manner. First, we conducted a genome-wide screen to survey genomic integration sites that support high transcriptional activity. We showed that CRISPR/Cas9-mediated de novo integration into one of these transcriptional hot-spots at the GRIK1 locus resulted in a 2.4-fold increase in heterologous gene expression over random integration. Subsequently, we set the groundwork necessary to evaluate a cell line development strategy that aims to increase the frequency of successful de novo targeted integrations. Second, we utilized two approaches for rational promoter engineering. We established a transcriptomics-guided workflow for de novo synthetic promoter design based on the Design-Build-Test paradigm. By using this workflow, we generated two synthetic designs that were comparable to a strong viral promoter and a strong endogenous promoter. We also employed an alternative approach by creating hybrid promoters, which resulted in a hybrid promoter variant that was also comparable to the same viral and endogenous promoters. Third, we exploited the general mammalian terminator structure and created a synthetic terminator that was comparable to a strong viral terminator. We evaluated 12 endogenous and 30 synthetic terminators for heterologous gene expression and revealed interactions between several key components of the terminator. Critically, we showed that transgene expression was 1.9x higher with endogenous and synthetic elements when compared with strong viral-derived elements. Ultimately, we showed that transgene expression can be finely adjusted by the approaches and methods described in this dissertation, and that viral-derived elements can be readily substituted by our synthetic designs